Identification of Secondary Nucleation Inhibitors of Amyloid-β Aggregation by Cellular Selection of a SICLOPPS...

Alzheimer's disease is characterized by the accumulation of amyloid beta (Aβ) aggregates. Soluble oligomers Aβ oligomeric intermediates (AβOs) generated during aggregation are hypothesized to be a neurotoxic species. Many cyclic peptides have been developed to inhibit Aβ aggregation but primarily target Aβ monomers and fibrils; few cyclic peptides selectively recognize AβOs. We selected a library of >10⁷ cyclic peptides generated by the widely used split-intein mediated circular ligation of peptides and proteins (SICLOPPS) strategy for binders of AβOs. These selections identified cyclo-CRLISFF, which significantly delayed Aβ42 aggregation in vitro but displayed a mechanism inconsistent with inhibitors selectively targeting AβOs. To resolve this discrepancy, we tested whether intermediates formed during SICLOPPS cyclic peptide generation might also possess AβO binding activity. Our experiments showed that the CRLISFF sequence was active as an intein-bound intermediate which selectively targeted AβOs by inhibiting the secondary nucleation step of the Aβ42 aggregation cascade. This intermediate has not been previously examined in studies employing SICLOPPS and may present a convoluting factor when using this technology to generate cyclic peptide libraries. The CRLISFF motif also retained activity when transplanted onto an unrelated protein scaffold, suggesting that SICLOPPS sequences may be compatible with peptide grafting strategies used to create protein-based binders.